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. 2017 Nov 21;2(11):8308–8312. doi: 10.1021/acsomega.7b01288

Circular Dichroistic Impacts of 1-(3-Dimethylaminopropyl)-3-ethylurea: Secondary Structure Artifacts Arising from Bioconjugation Using 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide

Matthew B Kubilius 1, Raymond S Tu 1,*
PMCID: PMC6645161  PMID: 31457370

Abstract

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1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) is a commonly used reagent for bioconjugation and peptide synthesis. Both EDC and the corresponding urea derivative, 1-(3-dimethylaminopropyl)-3-ethylurea (EDU), are achiral. As the reagent is active in aqueous solutions, it is a common choice for the study of evolving secondary structural changes via circular dichroism. This work highlights the effect of EDU on spectropolarimetric measurements, namely, the problematic absorption profile at low wavelengths (190–220 nm). We demonstrate that EDU is capable of erroneously indicating structural changes, particularly loss of α-helical character, through masking of the characteristic minimum at 208 nm. However, if the concentrations of the EDU in the sample are known, then this effect can be anticipated and calculations of secondary structure can be adjusted to avoid the impacted wavelengths. Impacts of EDU in a sample are compared to those of standard urea, which, by contrast, is commonly used as a denaturant in circular dichroism studies without issue.

Introduction

This work highlights the influence of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU) in circular dichroism (CD) measurements. The importance of this work stems from the ubiquity of the carbodiimide coupling agent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) in bioconjugation.1−5 The presence of EDU in CD samples can cause the underlying data to be obscured, preventing the interpretation of structure for protein and peptide bioconjugates. A key advantage of EDC bioconjugation is its compatibility with aqueous solutions, making its conjugation chemistry an attractive choice for study of evolving secondary structural changes using circular dichroism.6,7 For example, recent work has demonstrated a desire to study the self-assemblies of small (three amino acid) peptide sequences computationally and experimentally.8 With an understanding of the influence of the linking reagents on these measurements, these self-assemblies have the potential to be measured as they are grown.

EDC is used as a linking reagent for condensation reactions linking amines to carboxylic acid groups, typically in aqueous media. The EDU product of this reaction is the urea form of the original carbodiimide molecule, as shown in Figure 1. Because of the molecule’s ability to react with both water and the molecule of interest, EDC usage protocols generally recommend a 10:1 molar ratio of EDC to each carboxylic acid, yielding these same elevated concentrations of EDU in solution post-reaction.9

Figure 1.

Figure 1

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Reaction of EDC to EDU, either through use as a linking reagent, or via direct reaction with the aqueous solution.

Both EDC and EDU are achiral, and they might be assumed not to directly impact CD measurement. Unlike urea, however, EDU’s applicable concentration range, absorption impact, and wavelength range of interference on circular dichroism measurements are not commonly known. Establishing those parameters will allow for the development of well-controlled studies with clearly understood results using this reagent in solution. Typically, the issues explored in this work are eliminated in advance of structural studies by chromatography purification of samples post-conjugation. Although that method is undeniably effective, it is incompatible with kinetic studies or attempts to directly measure the change in the secondary structure as a function of reaction completion in real time.

We characterize the effect of EDU in a solution by direct inspection of CD signals of the common reference samples bovine serum albumin (BSA) and pantolactone measured in increasing concentrations of both urea and EDU. By evaluating the two-component system, we quantify the degree that standard urea can be used without issue, whereas the EDU can be problematic over similar concentration ranges. Because circular dichroism measures the difference in absorbance of left- and right-polarized light,10−13 any components in a solution able to change this ratio will give rise to changes in the spectra obtained. In this work, we highlight the degree that EDU absorbs over a much broader range of wavelengths as well as the impact that this adsorption has on secondary structure interpretation. Because only chiral molecules or molecules with certain fixed orientations give rise to such a difference, small, achiral molecules do not generally impact circular dichroism measurements. However, if an achiral species is present in a sample that significantly absorbs unpolarized light at the wavelength of the CD scan, it will absorb a greater fraction of the transmitted light, simply due to the greater availability of the light less absorbed by the chiral molecule. This absorption results in a reduction in the magnitude of the reported circular dichroism measurements because of the disproportionate absolute absorption.

Results and Discussion

To quantify the nature of EDU’s effect on a system, a series of circular dichroism spectra are obtained on bovine serum albumin (BSA) in deionized (DI) water (Figure 2). With no EDU present, the protein shows a clear α-helical character. But at EDU concentrations as low as 0.05 M, the characteristic minimum expected near 208 nm is completely obscured. As EDU, being achiral, has a zero-magnitude CD signal of its own (Supporting Information), this effect cannot be thought of as the summation of the CD spectra of the two molecules. At 0.1 M EDU, the minimum at 222 has shifted to a higher wavelength and noticeably decreased in intensity. This shift continues with increasing concentration until a sample concentration of 0.4 M EDU, at which point it is no longer possible to observe the helical signal associated with BSA. Note that the signal decays to zero with increasing EDU concentration. As we see signal loss, rather than signal transition to a random coil configuration, we establish that the effect is due to EDU’s impact on the CD signal, as opposed to EDU denaturing the secondary structure of the protein molecules.

Figure 2.

Figure 2

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CD spectra of BSA at various concentrations of 1-(3-dimethylaminopropyl)-3-ethylurea.

To better understand the magnitude of this phenomenon, we perform the same experiment using urea (Figure 3). A small loss is seen around the 210 nm wavelength, but the signal persists. Urea is a known denaturant, and the purpose of Figure 3 is to highlight the relative intensity of the data in Figure 2. Given that denaturation is potentially a factor in the EDU results, we performed a second experiment in which the signal loss could be decoupled from secondary structure changes.

Figure 3.

Figure 3

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CD spectra of BSA at various concentrations of urea.

We duplicated the CD experiments with (S)-(+)-pantolactone replacing the BSA (Figure 4). (S)-(+)-Pantolactone is a chiral molecule incapable of secondary structure-based signal loss,14 and it is often used as a circular dichroism standard with a known minimum at 219 nm. Neither urea nor EDU will have a structural effect on this molecule, and their effects on absorbance and CD signal can be decoupled from losses in the secondary structure.

Figure 4.

Figure 4

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1-(3-Dimethylaminopropyl)-3-ethylurea interferes with pantolactone circular dichroism spectra as a function of EDU concentration. Inset: CD spectra of pantolactone at various concentrations of urea.

When combining (S)-(+)-pantolactone with EDU (Figure 4), a clear trend of complete data obfuscation is seen, analogous to that seen in Figure 2. From this, we conclude that EDU’s absorbance gives rise to these results, as opposed to structural changes arising from the EDU acting as a denaturant.

The two-component system with pantolactone and urea corroborates the hypothesis (Figure 4, inset). At 0.4 M urea, only minor changes in the data are seen, reinforcing the notion that urea does not interfere with circular dichroism measurements at these wavelengths and concentrations. The effects of urea are seen in the wavelengths below 200 nm, which is consistent with the standard practice of anticipating urea effects in data below 210 nm. From this, we can infer that the subtle changes seen in Figure 3 centered on 210 nm are a result of urea-induced denaturation of α-helical structures into random coils.

Figure 5 quantifies the mechanism behind these results, plotting the absorbance data of 0.1 M EDU, 0.1 M urea, and 1 mg/mL bovine serum albumin (BSA) samples from 200 to 250 nm in a 1 cm quartz cuvette. Concentrations and units were chosen to match those typically studied in circular dichroism experiments. Urea is seen with its characteristic high absorbance at low wavelengths, preventing reliable data collection below 210 nm. EDU, by contrast, absorbs heavily over the entire range of interest. Although this molecule is capable of impacting readings over the entire range, note that the error would be gradually, increasingly prominent at lower wavelengths. This results in the EDU yielding circular dichroism errors even at concentrations well below those deemed acceptable for urea.

Figure 5.

Figure 5

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Absorbance profiles of 0.1 M EDU (red circles), 0.1 M urea (blue triangles), and 1 mg/mL bovine serum albumin (black squares) measured in a 1 cm quartz cuvette.

From these results, we suggest a new “rule of thumb” for the use of EDC in bioconjugation when paired with circular dichroism. At low (<0.05 M) concentrations, EDU can be present with minimal effect. Wavelengths above 215 nm remain largely unaffected at concentrations encountered in the common practice of single-molecule PEGylations of a protein.15,16 By contrast, “EDC-heavy” syntheses, where the concentration of EDU is orders of magnitude higher than the species of interest, would be incompatible with this molecule due to signal loss.

At moderate solution concentrations (0–0.2 M), thoughtful choice of wavelength ranges can be used to avoid the issue. Signal loss increases from low wavelengths to high as a function of concentration. Because the pattern is easily seen, signal loss thresholds from Figures 3 and 4 can be collapsed onto a single curve to generate a plot of the lowest suggested wavelength to be taken into consideration during secondary structure calculations as a function of EDC/EDU concentration (Figure 6). This is analogous to the existing practice of only considering wavelengths higher than 210 nm for samples in urea solutions. As an example, in a solution of 0.1 M EDU, secondary structure calculations should use CD data only at wavelengths of 225 nm and above.

Figure 6.

Figure 6

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Minimum wavelength at which a given EDU concentration does not cause significant signal loss at a 1 mm path length.

To keep signal-to-noise ratios high in such circumstances, one should keep the total absorbance (−log I/Io) of the sample low, typically below 1.0. Previous work has suggested that there is a theoretical maximum in signal-to-noise ratio at an overall sample absorbance of 0.869.11,17 This overall absorbance includes the additives included in the sample. Common additives for protein systems include urea at concentrations as high as 8 M.18,19 The ability to use such high concentrations of urea is possible because the protein secondary structure characterization is evaluated at wavelengths between 205 and 240 nm, and 8 M urea absorbs strongly below 210 nm. Therefore, using urea as a denaturant, structural changes inferred from signals at 222 or 225 nm are not significantly obscured.

Experimental Procedures

A stock solution of EDU was purchased from Sigma-Aldrich (E7750-10G, Lot #SLBH9924V) and prepared by allowing a solution of the desired final molar concentration of EDC in water to hydrolyze completely into the urea form by standing overnight. (R)-(−)-Pantolactone 99% was purchased from Aldrich (237817-5G, Lot #07115TB) and dissolved in DI water as a stock solution. Similarly, albumin, monomer bovine was purchased from Sigma (A1900-250MG, Lot #031M7407V) and dissolved in 1.0 M sodium chloride (Fisher, S271-3) to form the helical state in solution. Sample solutions were prepared by combining these stock solutions in additional DI/1.0 M NaCl solutions to arrive at the desired final molar concentrations. Absorbance measurements were taken in 1 cm path length quartz cuvettes (Starna 21-Q-1) on an OLIS DSM20 in the absorbance mode. Circular dichroism measurements were made using a 1 mm path length quartz sample cell (Starna 31-Q-1) on the OLIS DSM20 in a circular dichroism mode.

Acknowledgments

We would like to thank NSF grant #1006407 and AFOSR grant #FA9550-14-1-0263 for the financial support of this work. M.B.K. would like to thank MSRDC grant #D01_W911SR-14-2-0001-0001 for partial support of this work.

Glossary

Abbreviations

CD

circular dichroism

BSA

bovine serum albumin

EDC

1-ethyl-3-[3-dimethylaminopropyl]carbodiimide

EDU

1-(3-dimethylaminopropyl)-3-ethylurea

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01288.

  • Circular dichroism of EDU in deionized water; circular dichroism of a PEGylation bioconjugation showing accumulation of EDU (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao7b01288_si_001.pdf (362.5KB, pdf)

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Supplementary Materials

ao7b01288_si_001.pdf (362.5KB, pdf)

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